Root-preferred promoter and methods of use

ABSTRACT

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for a promoter for the gene encoding  Sorghum bicolor  RCc3. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant or plant cell with a nucleotide sequence operably linked to one of the promoters of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/442,930, filed Feb. 15, 2011, which is hereby incorporated herein inits entirety by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of an operably linked promoter that is functionalwithin the plant host. Choice of the promoter sequence will determinewhen and where within the organism the heterologous DNA sequence isexpressed. Where expression in specific tissues or organs is desired,tissue-preferred promoters may be used. Where gene expression inresponse to a stimulus is desired, inducible promoters are theregulatory element of choice. In contrast, where continuous expressionis desired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in the expressionconstructs of transformation vectors to bring about varying levels ofexpression of heterologous nucleotide sequences in a transgenic plant.

Frequently it is desirable to express a DNA sequence in particulartissues or organs of a plant. For example, increased resistance of aplant to infection by soil- and air-borne pathogens might beaccomplished by genetic manipulation of the plant's genome to comprise atissue-preferred promoter operably linked to a heterologouspathogen-resistance gene such that pathogen-resistance proteins areproduced in the desired plant tissue.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a tissue-preferred promoter operably linked to anantisense nucleotide sequence, such that expression of the antisensesequence produces an RNA transcript that interferes with translation ofthe mRNA of the native DNA sequence.

Thus far, the regulation of gene expression in plant roots has not beenadequately studied despite the importance of the root to plantdevelopment. To some degree this is attributable to a lack of readilyavailable, root-specific biochemical functions whose genes may becloned, studied, and manipulated. Genetically altering plants throughthe use of genetic engineering techniques and thus producing a plantwith useful traits requires the availability of a variety of promoters.An accumulation of promoters would enable the investigator to designrecombinant DNA molecules that are capable of being expressed at desiredlevels and cellular locales. Therefore, a collection of tissue-preferredpromoters would allow for a new trait to be expressed in the desiredtissue.

Thus, isolation and characterization of tissue-preferred, particularlyroot-preferred, promoters that can serve as regulatory regions forexpression of heterologous nucleotide sequences of interest in atissue-preferred manner are needed for genetic manipulation of plants.

SUMMARY OF THE INVENTION

Compositions and methods for regulating expression of a heterologousnucleotide sequence of interest in a plant or plant cell are provided.Compositions comprise novel nucleotide sequences for promoters thatinitiate transcription. Embodiments of the invention comprise thenucleotide sequence set forth in SEQ ID NO: 1 or a complement thereof,the nucleotide sequence comprising the plant promoter sequence of theplasmid deposited as Patent Deposit No. NRRL B-50462 or a complementthereof, a nucleotide sequence comprising at least 20 contiguousnucleotides of SEQ ID NO: 1, wherein said sequence initiatestranscription in a plant cell, and a nucleotide sequence comprising asequence having at least 85% sequence identity to the sequence set forthin SEQ ID NO: 1, wherein said sequence initiates transcription in theplant cell.

A method for expressing a heterologous nucleotide sequence in a plant orplant cell is provided. The method comprises introducing into a plant ora plant cell an expression cassette comprising a heterologous nucleotidesequence of interest operably linked to one of the promoters of thepresent invention. In this manner, the promoter sequences are useful forcontrolling the expression of the operably linked heterologousnucleotide sequence. In specific methods, the heterologous nucleotidesequence of interest is expressed in a root-preferred manner.

Further provided is a method for expressing a nucleotide sequence ofinterest in a root-preferred manner in a plant. The method comprisesintroducing into a plant cell an expression cassette comprising apromoter of the invention operably linked to a heterologous nucleotidesequence of interest.

Expression of the nucleotide sequence of interest can provide formodification of the phenotype of the plant. Such modification includesmodulating the production of an endogenous product, as to amount,relative distribution, or the like, or production of an exogenousexpression product to provide for a novel function or product in theplant. In specific methods and compositions, the heterologous nucleotidesequence of interest comprises a gene product that confers herbicideresistance, pathogen resistance, insect resistance, and/or alteredtolerance to salt, cold, or drought.

Expression cassettes comprising the promoter sequences of the inventionoperably linked to a heterologous nucleotide sequence of interest areprovided. Additionally provided are transformed plant cells, planttissues, seeds, and plants.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods drawn to plantpromoters and methods of their use. The compositions comprise nucleotidesequences for the promoter region of a sorghum (Sorghum bicolor) genewith strong similarity to RCc3 in rice, which was reported to beexpressed in a root specific manner (Xu Y, et al. (1995) Plant Mol.Biol. 27: 237-248. The compositions further comprise DNA constructscomprising a nucleotide sequence for the promoter region of the sorghumRCc3 gene operably linked to a heterologous nucleotide sequence ofinterest. Accordingly, the promoter set forth in SEQ ID NO: 1 was giventhe identifying name “Sb-RCc3 promoter.” In particular, the presentinvention provides for isolated nucleic acid molecules comprising thenucleotide sequence set forth in SEQ ID NO: 1, and the plant promotersequence deposited in bacterial hosts as Patent Deposit No. NRRLB-50462, on Jan. 25, 2011, and fragments, variants, and complementsthereof.

Plasmids containing the plant promoter nucleotide sequences of theinvention were deposited on Jan. 25, 2011 with the Patent Depository ofthe Agricultural Research Service Culture Collection of the NationalCenter for Agricultural Utilization Research, at 1815N. UniversityStreet, Peoria, Ill., 61604, and assigned Patent Deposit No. NRRLB-50462. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112. The deposit will irrevocablyand without restriction or condition be available to the public uponissuance of a patent. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

The Sb-RCc3 promoter sequences of the present invention includenucleotide constructs that allow initiation of transcription in a plant.In specific embodiments, the Sb-RCc3 promoter sequence allows initiationof transcription in a tissue-preferred, more particularly in aroot-preferred manner. Such constructs of the invention compriseregulated transcription initiation regions associated with plantdevelopmental regulation. Thus, the compositions of the presentinvention include DNA constructs comprising a nucleotide sequence ofinterest operably linked to the Sb-RCc3 promoter sequence. The sequencefor the Sb-RCc3 promoter region is set forth in SEQ ID NO: 1.

Compositions of the invention include the nucleotide sequences for thenative Sb-RCc3 promoter and fragments and variants thereof. In specificembodiments, the promoter sequences of the invention are useful forexpressing sequences of interest in a tissue-preferred, particularly aroot-preferred manner. The nucleotide sequences of the invention alsofind use in the construction of expression vectors for subsequentexpression of a heterologous nucleotide sequence in a plant of interestor as probes for the isolation of other Sb-RCc3-like promoters.

The invention encompasses isolated or substantially purified nucleicacid compositions. An “isolated” or “purified” nucleic acid molecule orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acid isfree of sequences (optimally protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. The Sb-RCc3 promoter sequences of theinvention may be isolated from the 5′ untranslated region flanking theirrespective transcription initiation sites.

Fragments and variants of the disclosed promoter nucleotide sequencesare also encompassed by the present invention. In particular, fragmentsand variants of the Sb-RCc3 promoter sequence of SEQ ID NO: 1 may beused in the DNA constructs of the invention. As used herein, the term“fragment” refers to a portion of the nucleic acid sequence. Fragmentsof an Sb-RCc3 promoter sequence may retain the biological activity ofinitiating transcription, more particularly driving transcription in aroot-preferred manner. Alternatively, fragments of a nucleotide sequencewhich are useful as hybridization probes may not necessarily retainbiological activity. Fragments of a nucleotide sequence for the promoterregion of the Sb-RCc3 gene may range from at least about 20 nucleotides,about 50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence of the invention for the promoter region of thegene.

A biologically active portion of an Sb-RCc3 promoter can be prepared byisolating a portion of the Sb-RCc3 promoter sequence of the invention,and assessing the promoter activity of the portion. Nucleic acidmolecules that are fragments of an Sb-RCc3 promoter nucleotide sequencecomprise at least about 16, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1550, 1600, 1650, or 1700 nucleotides, or up to the number ofnucleotides present in a full-length Sb-RCc3 promoter sequence disclosedherein (for example, 1710 nucleotides for SEQ ID NO: 1).

As used herein, the term “variants” means substantially similarsequences. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, suchas, for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein.

For nucleotide sequences, a variant comprises a deletion and/or additionof one or more nucleotides at one or more internal sites within thenative polynucleotide and/or a substitution of one or more nucleotidesat one or more sites in the native polynucleotide. As used herein, a“native” nucleotide sequence comprises a naturally occurring nucleotidesequence. For nucleotide sequences, naturally occurring variants can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a nucleotide sequence of the invention may differ fromthat sequence by as few as 1-15 nucleic acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 nucleic acidresidue.

Variant nucleotide sequences also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. With sucha procedure, Sb-RCc3 nucleotide sequences can be manipulated to create anew Sb-RCc3 promoter. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire Sb-RCc3 sequenceset forth herein or to fragments thereof are encompassed by the presentinvention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from genomic DNAextracted from any plant of interest. Methods for designing PCR primersand PCR cloning are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafterSambrook. See also Innis et al., eds. (1990) PCR Protocols: A Guide toMethods and Applications (Academic Press, New York); Innis and Gelfand,eds. (1995) PCR Strategies (Academic Press, New York); and Innis andGelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).Known methods of PCR include, but are not limited to, methods usingpaired primers, nested primers, single specific primers, degenerateprimers, gene-specific primers, vector-specific primers,partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments from a chosen organism. The hybridization probes may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the Sb-RCc3 promotersequence of the invention. Methods for preparation of probes forhybridization and for construction of genomic libraries are generallyknown in the art and are disclosed in Sambrook.

For example, the entire Sb-RCc3 promoter sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding Sb-RCc3 promote sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among Sb-RCc3promoter sequence and are at least about 10 nucleotides in length or atleast about 20 nucleotides in length. Such probes may be used to amplifycorresponding Sb-RCc3 promoter sequence from a chosen plant by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism, or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook.

Hybridization of such sequences may be carried out under stringentconditions. The terms “stringent conditions” or “stringent hybridizationconditions” are intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for a duration of atleast 30 minutes. Duration of hybridization is generally less than about24 hours, usually about 4 to about 12 hours. The duration of the washtime will be at least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) (thermal melting point)can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See also Sambrook.

Thus, isolated sequences that have root-preferred promoter activity andwhich hybridize under stringent conditions to the Sb-RCc3 promotersequences disclosed herein, or to fragments thereof, are encompassed bythe present invention.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et at (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seethe website for the National Center for Biotechnology Information.Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. An“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, optimally at least 80%, more optimally at least 90%,and most optimally at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species include corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mung bean, lima bean, fava bean, lentils,chickpea, etc.

Heterologous coding sequences expressed by the Sb-RCc3 promoters of theinvention may be used for varying the phenotype of a plant. Variouschanges in phenotype are of interest including modifying expression of agene in a plant root, altering a plant's pathogen or insect defensemechanism, increasing the plants tolerance to herbicides in a plant,altering root development to respond to environmental stress, modulatingthe plant's response to salt, temperature (hot and cold), drought, andthe like. These results can be achieved by the expression of aheterologous nucleotide sequence of interest comprising an appropriategene product. In specific embodiments, the heterologous nucleotidesequence of interest is an endogenous plant sequence whose expressionlevel is increased in the plant or plant part. Alternatively, theresults can be achieved by providing for a reduction of expression ofone or more endogenous gene products, particularly enzymes,transporters, or cofactors, or by affecting nutrient uptake in theplant. These changes result in a change in phenotype of the transformedplant.

General categories of nucleotide sequences of interest for the presentinvention include, for example, those genes involved in information,such as zinc fingers, those involved in communication, such as kinases,and those involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes, for example, include genes encodingimportant traits for agronomics, insect resistance, disease resistance,herbicide resistance, and environmental stress resistance (alteredtolerance to cold, salt, drought, etc). It is recognized that any geneof interest can be operably linked to the promoter of the invention andexpressed in the plant.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European corn borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as those which detoxify fumonisin (U.S. Pat. No. 5,792,931);avirulence (avr) and disease resistance (R) genes (Jones et al. (1994)Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos etal. (1994) Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene),glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360) or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Glyphosate resistance is imparted by mutant 5-enolpyruvl-3-phosphikimatesynthase (EPSP) and aroA genes. See, for example, U.S. Pat. No.4,940,835 to Shah et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes.See also U.S. Pat. Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and internationalpublications WO 97/04103; WO 97/04114; WO 00/66746; WO 01/66704; WO00/66747 and WO 00/66748, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene that encodes a glyphosate oxido-reductase enzyme asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, whichare incorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,714,188 and 7,462,481.

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like.

Examples of other applicable genes and their associated phenotypeinclude the gene which encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that promote yield improvement; and genes thatprovide for resistance to stress, such as cold, dehydration resultingfrom drought, heat and salinity, toxic metal or trace elements, or thelike.

As noted, the heterologous nucleotide sequence operably linked to theSb-RCc3 promoter disclosed herein may be an antisense sequence for atargeted gene. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant root.

“RNAi” refers to a series of related techniques to reduce the expressionof genes (See for example U.S. Pat. No. 6,506,559). Older techniquesreferred to by other names are now thought to rely on the samemechanism, but are given different names in the literature. Theseinclude “antisense inhibition,” the production of antisense RNAtranscripts capable of suppressing the expression of the target protein,and “co-suppression” or “sense-suppression,” which refer to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020, incorporated herein by reference). Suchtechniques rely on the use of constructs resulting in the accumulationof double stranded RNA with one strand complementary to the target geneto be silenced. The Sb-RCc3 promoter of the embodiments may be used todrive expression of constructs that will result in RNA interferenceincluding microRNAs and siRNAs.

As used herein, the terms “promoter” or “transcriptional initiationregion” mean a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter may additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter regions disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe 5′ untranslated region upstream from the particular promoter regionsidentified herein. Additionally, chimeric promoters may be provided.Such chimeras include portions of the promoter sequence fused tofragments and/or variants of heterologous transcriptional regulatoryregions. Thus, the promoter regions disclosed herein can compriseupstream regulatory elements such as, those responsible for tissue andtemporal expression of the coding sequence, enhancers and the like. Inthe same manner, the promoter elements, which enable expression in thedesired tissue such as the root, can be identified, isolated and usedwith other core promoters to confer root-preferred expression. In thisaspect of the invention, “core promoter” is intended to mean a promoterwithout promoter elements.

In the context of this disclosure, the term “regulatory element” alsorefers to a sequence of DNA, usually, but not always, upstream (5′) tothe coding sequence of a structural gene, which includes sequences whichcontrol the expression of the coding region by providing the recognitionfor RNA polymerase and/or other factors required for transcription tostart at a particular site. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.A promoter element comprises a core promoter element, responsible forthe initiation of transcription, as well as other regulatory elements(as discussed elsewhere in this application) that modify geneexpression. It is to be understood that nucleotide sequences, locatedwithin introns, or 3′ of the coding region sequence may also contributeto the regulation of expression of a coding region of interest. Examplesof suitable introns include, but are not limited to, the maize IVS6intron, or the maize actin intron. A regulatory element may also includethose elements located downstream (3′) to the site of transcriptioninitiation, or within transcribed regions, or both. In the context ofthe present invention a post-transcriptional regulatory element mayinclude elements that are active following transcription initiation, forexample translational and transcriptional enhancers, translational andtranscriptional repressors, and mRNA stability determinants.

The regulatory elements, or variants or fragments thereof, of thepresent invention may be operatively associated with heterologousregulatory elements or promoters in order to modulate the activity ofthe heterologous regulatory element. Such modulation includes enhancingor repressing transcriptional activity of the heterologous regulatoryelement, modulating post-transcriptional events, or either enhancing orrepressing transcriptional activity of the heterologous regulatoryelement and modulating post-transcriptional events. For example, one ormore regulatory elements, or fragments thereof, of the present inventionmay be operatively associated with constitutive, inducible, or tissuespecific promoters or fragment thereof, to modulate the activity of suchpromoters within desired tissues in plant cells.

The regulatory sequences of the present invention, or variants orfragments thereof, when operably linked to a heterologous nucleotidesequence of interest can drive root-preferred expression of theheterologous nucleotide sequence in the root (or root part) of the plantexpressing this construct. The term “root-preferred,” means thatexpression of the heterologous nucleotide sequence is most abundant inthe root or a root part, including, for example, the root cap, apicalmeristem, protoderm, ground meristem, procambium, endodermis, cortex,vascular cortex, epidermis, and the like. While some level of expressionof the heterologous nucleotide sequence may occur in other plant tissuetypes, expression occurs most abundantly in the root or root part,including primary, lateral and adventitious roots.

A “heterologous nucleotide sequence” is a sequence that is not naturallyoccurring with the promoter sequence of the invention. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.

The isolated promoter sequences of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter region may beutilized and the ability to drive expression of the nucleotide sequenceof interest retained. It is recognized that expression levels of themRNA may be altered in different ways with deletions of portions of thepromoter sequences. The mRNA expression levels may be decreased, oralternatively, expression may be increased as a result of promoterdeletions if, for example, there is a negative regulatory element (for arepressor) that is removed during the truncation process. Generally, atleast about 20 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence.

It is recognized that to increase transcription levels, enhancers may beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like. Someenhancers are also known to alter normal promoter expression patterns,for example, by causing a promoter to be expressed constitutively whenwithout the enhancer, the same promoter is expressed only in onespecific tissue or a few specific tissues.

Modifications of the isolated promoter sequences of the presentinvention can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, a “weak promoter” means a promoter thatdrives expression of a coding sequence at a low level. A “low level” ofexpression is intended to mean expression at levels of about 1/10,000transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts.

It is recognized that the promoters of the invention may be used withtheir native Sb-RCc3 coding sequences to increase or decreaseexpression, thereby resulting in a change in phenotype of thetransformed plant. This phenotypic change could further affect anincrease or decrease in levels of metal ions in tissues of thetransformed plant.

The nucleotide sequences disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant. The Sb-RCc3 promoter sequence is useful in this aspectwhen operably linked with a heterologous nucleotide sequence whoseexpression is to be controlled to achieve a desired phenotypic response.The term “operably linked” means that the transcription or translationof the heterologous nucleotide sequence is under the influence of thepromoter sequence. In this manner, the nucleotide sequences for thepromoters of the invention may be provided in expression cassettes alongwith heterologous nucleotide sequences of interest for expression in theplant of interest, more particularly for expression in the root of theplant.

Such expression cassettes will comprise a transcriptional initiationregion comprising one of the promoter nucleotide sequences of thepresent invention, or variants or fragments thereof, operably linked tothe heterologous nucleotide sequence. Such an expression cassette can beprovided with a plurality of restriction sites for insertion of thenucleotide sequence to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes as well as 3′ termination regions.

The expression cassette can include, in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter, orvariant or fragment thereof, of the invention), a translationalinitiation region, a heterologous nucleotide sequence of interest, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

While it may be preferable to express a heterologous nucleotide sequenceusing the promoters of the invention, the native sequences may beexpressed. Such constructs would change expression levels of the Sb-RCc3protein in the plant or plant cell. Thus, the phenotype of the plant orplant cell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence beingexpressed, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

The expression cassette comprising the sequences of the presentinvention may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) can be provided on another expression cassette.

Where appropriate, the nucleotide sequences whose expression is to beunder the control of the root-preferred promoter sequence of the presentinvention and any additional nucleotide sequence(s) may be optimized forincreased expression in the transformed plant. That is, these nucleotidesequences can be synthesized using plant preferred codons for improvedexpression. See, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11 for a discussion of host-preferred codon usage. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989)Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allisonet al. (1986) Virology 154:9-20); MDMV leader (Maize Dwarf MosaicVirus); human immunoglobulin heavy-chain binding protein (BiP) (Macejaket al. (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987)Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al.(1989) Molecular Biology of RNA, pages 237-256); and maize chloroticmottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385).See also Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Methodsknown to enhance mRNA stability can also be utilized, for example,introns, such as the maize Ubiquitin intron (Christensen and Quail(1996) Transgenic Res. 5:213-218; Christensen et al. (1992) PlantMolecular Biology 18:675-689) or the maize AdhI intron (Kyozuka et al.(1991) Mol. Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica35:353-357), and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

Reporter genes or selectable marker genes may be included in theexpression cassettes. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737;Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991)Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) PlantMol. Biol. 5:103-108; and Zhijian et al. (1995) Plant Science108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet.210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) TransgenicRes. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423);glyphosate (Shaw et al. (1986) Science 233:478-481; and U.S. applicationSer. Nos. 10/004,357; and 10/427,692); phosphinothricin (DeBlock et al.(1987) EMBO J. 6:2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (beta-glucuronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescence protein;Chalfie et al. (1994) Science 263:802), luciferase (Riggs et al. (1987)Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992) MethodsEnzymol. 216:397-414) and the maize genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247:449).

The expression cassette comprising the Sb-RCc3 promoter of the presentinvention operably linked to a nucleotide sequence of interest can beused to transform any plant. In this manner, genetically modifiedplants, plant cells, plant tissue, seed, root, and the like can beobtained.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055 and Zhao et al., U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the DNA constructs comprising the promotersequences of the invention can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, viral vector systems and theprecipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, the transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use particles coated with polyethylimine (PEI; Sigma#P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into its genome.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Identification of Sb-RCc3 Gene

The Sb-RCc3 gene was identified through a search of expression profilingdata obtained from the elite inbred line BTX623. Tissue from greenhousegrown plants was sampled from each of the major organs at a 6 leafvegetative stage and in late bloom stage (just prior to pollen shed).Tissue from reproductive organs was also collected. Three replicateswere taken for each sample, with each replicate consisting of nineplants. RNA was isolated from each of the replicates, reversetranscribed, and sequenced using Solexa DNA sequencing technology(Illumina). Sequence “tags” were aligned with publically availablegenomic sequence to identify the gene. A comparison of expression ineach of the samples identified genes with root-preferred expression.

Example 2 PCR Isolation of the Sb-RCc3 Promoter

Once a gene was identified, 5′ flanking sequence was obtained bysearching publically available genomic data for sequence that was 5′ ofthe coding region. For Sb-RCc3, approximately 1710 bp of upstreamsequence was identified. A NotI restriction endonuclease recognitionsite was added to the 5′ end of the sequence and a BamH1 recognitionsite was added to the 3′ end of the sequence to facilitate ligation ofthe DNA into an expression vector once it was chemically synthesized.Analysis of the sequence for motifs revealed a putative TATA boxapproximately 108 bp from the 3′ end of the sequence and a putativetranscription start site approximately 83 bp from the 3′ end.

Example 3 Expression Analysis in Transgenic Maize Plants

Stable transformed plants were created using Agrobacterium protocols(detailed in Example 4) to allow for characterization of promoteractivity, including expression pattern and expression level directed bythe promoter. The Sb-RCc3 promoter (SEQ ID NO: 1) was operably connectedto either the B-glucuronidase (GUS) gene (abbreviated as Sb-RCc3:GUS) oran insecticidal gene (abbreviated Sb-RCc3:IG1). The promoter was alsooperably linked to the Adh1 intron (intron 1) and IG1 gene (abbreviatedas Sb-RCc3(Adh1 intron1):IG1) for the purpose of potentially increasingexpression as it has been shown that in cereal plant cells theexpression of transgenes is enhanced by the presence of some 5′ proximalintrons (See Callis et al. (1987) Genes and Development 1: 1183-1200;Kyozuka et al. (1990) Maydica 35:353-357). The use of the GUS geneallowed the expression pattern directed by the promoter to be visualizedby histochemically staining tissue for GUS enzymatic activity.

Twenty-six GUS events were regenerated and grown under greenhouseconditions until they reached a growth stage ranging from V4 to V6.Vegetative growth stages are determined by the number of collared leaveson the plant. Therefore, a plant at V6 stage has 6 fully collaredleaves. Leaf and root tissue were sampled from each plant at this stage.The plants were then allowed to grow to early R1 stage, a point justprior to pollen shed, where silk, stalk, and tassel tissue werecollected. Finally, pollen was collected when the plants startedshedding.

Results from Sb-RCc3:GUS showed that the Sb-RCc3 promoter droveexpression in maize roots. Expression was detected in the mature regionsof the root, primarily in the cortex. In the root tip, expression wasdetected in the elongation region, but not in the meristem or in theroot cap. Expression was not detected in leaf, tassel, or silk tissues.It also was not detected in pollen. Stalks did not show expression;however, in approximately half of the events expression was detected inthe vasculature of the sheath. The expression was not strong relative tothe expression level in roots.

TABLE 1 Maize Expression Results¹ for Sb-RCc3: GUS V5-V6 R1-R2 Leaf RootStalk Tassel Silk Pollen Sb-RCc3 0 2 0 0 0 0 Ubi-1 2 3 3 3 2 3Untransformed 0 0 0 0 0 0 (negative control) ¹Histochemical stainingdata is represented on a 0-3 scale with the well characterized maizeUbi-1 promoter serving as a reference point. The Ubi-1 promoter is astrong constitutive promoter in nearly all tissues of maize.

Twenty-four transgenic maize plants expressing Sb-RCc3:IG1 and 15 plantsexpressing Sb-RCc3:ADH intron:IG1 were evaluated in the greenhouse.Quantitative ELISA on root, which included root tip and mature tissuetogether, and leaf material showed expression occurred only in rootswith both vectors. This supported the observation made using GUS. TheADH intron was included for the purpose of increasing expression.However, expression using Sb-RCc3 was better without the ADH intron byabout 2.7 fold. Expression of Sb-RCc3:IG1 relative to the well-knownmaize ubiquitin promoter was about 2 fold less.

TABLE 2 Maize Expression Results¹ for Sb-RCc3 and IG1 V5-V6 Leaf RootSb-RCc3 0 2 Sb-RCc3:ADH 0 1 Ubi-1 2 3 Untransformed 0 0 (negativecontrol) ¹Histochemical staining data is represented on a 0-3 scale withthe well characterized maize Ubi-1 promoter serving as a referencepoint. The Ubi-1 promoter is a strong constitutive promoter in nearlyall tissues of maize.

Example 4 Transformation and Regeneration of Transgenic Plants UsingAgrobacterium Mediated Transformation

For Agrobacterium-mediated transformation of maize with an Sb-RCc3promoter sequence of the embodiments, the method of Zhao was employed(U.S. Pat. No. 5,981,840, (hereinafter the '840 patent) and PCT patentpublication WO98/32326; the contents of which are hereby incorporated byreference).

Agrobacterium were grown on a master plate of 800 medium and cultured at28° C. in the dark for 3 days, and thereafter stored at 4° C. for up toone month. Working plates of Agrobacterium were grown on 810 mediumplates and incubated in the dark at 28° C. for one to two days.

Briefly, embryos were dissected from fresh, sterilized corn ears andkept in 561Q medium until all required embryos were collected. Embryoswere then contacted with an Agrobacterium suspension prepared from theworking plate, in which the Agrobacterium contained a plasmid comprisingthe promoter sequence of the embodiments. The embryos were co-cultivatedwith the Agrobacterium on 562P plates, with the embryos placed axis downon the plates, as per the '840 patent protocol.

After one week on 562P medium, the embryos were transferred to 563Omedium. The embryos were subcultured on fresh 563O medium at 2 weekintervals and incubation was continued under the same conditions. Callusevents began to appear after 6 to 8 weeks on selection.

After the calli had reached the appropriate size, the calli werecultured on regeneration (288W) medium and kept in the dark for 2-3weeks to initiate plant regeneration. Following somatic embryomaturation, well-developed somatic embryos were transferred to mediumfor germination (272V) and transferred to a lighted culture room.Approximately 7-10 days later, developing plantlets were transferred to272V hormone-free medium in tubes for 7-10 days until plantlets werewell established. Plants were then transferred to inserts in flats(equivalent to 2.5″ pot) containing potting soil and grown for 1 week ina growth chamber, subsequently grown an additional 1-2 weeks in thegreenhouse, then transferred to classic 600 pots (1.6 gallon) and grownto maturity.

Media Used in Agrobacterium-Mediated Transformation and Regeneration ofTransgenic Maize Plants:

561Q medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 68.5g/L sucrose, 36.0 g/L glucose, 1.5 mg/L 2,4-D, and 0.69 g/L L-proline(brought to volume with dI H₂O following adjustment to pH 5.2 with KOH);2.0 g/L Gelrite™ (added after bringing to volume with dI H₂O); and 8.5mg/L silver nitrate (added after sterilizing the medium and cooling toroom temperature).

800 medium comprises 50.0 mL/L stock solution A and 850 mL dI H₂O, andbrought to volume minus 100 mL/L with dI H₂O, after which is added 9.0 gof phytagar. After sterilizing and cooling, 50.0 mL/L BAstock solution Bis added, along with 5.0 g of glucose and 2.0 mL of a 50 mg/mL stocksolution of spectinomycin. Stock solution A comprises 60.0 g of dibasicK₂HPO₄ and 20.0 g of monobasic sodium phosphate, dissolved in 950 mL ofwater, adjusted to pH 7.0 with KOH, and brought to 1.0 L volume with dIH₂O, Stock solution B comprises 20.0 g NH₄Cl, 6.0 g MgSO₄.7H₂O, 3.0 gpotassium chloride, 0.2 g CaCl₂, and 0.05 g of FeSO₄.7H₂O, all broughtto volume with dI H₂O, sterilized, and cooled.

810 medium comprises 5.0 g yeast extract (Difco), 10.0 g peptone(Difco), 5.0 g NaCl, dissolved in dI H₂O, and brought to volume afteradjusting pH to 6.8. 15.0 g of bacto-agar is then added, the solution issterilized and cooled, and 1.0 mL of a 50 mg/mL stock solution ofspectinomycin is added.

562P medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, and 2.0 mg/L 2,4-D (brought to volume with dI H₂O followingadjustment to pH 5.8 with KOH); 3.0 g/L Gelrite™ (added after bringingto volume with dI H₂O); and 0.85 mg/L silver nitrate and 1.0 mL of a 100mM stock of acetosyringone (both added after sterilizing the medium andcooling to room temperature).

563O medium comprises 4.0 g/L N6 basal salts (SIGMA C-1416), 1.0 mL/LEriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0g/L sucrose, 1.5 mg/L 2,4-D, 0.69 g L-proline, and 0.5 g MES buffer(brought to volume with dI H₂O following adjustment to pH 5.8 with KOH).Then, 6.0 g/L Ultrapure™ agar-agar (EM Science) is added and the mediumis sterilized and cooled. Subsequently, 0.85 mg/L silver nitrate, 3.0 mLof a 1 mg/mL stock of Bialaphos, and 2.0 mL of a 50 mg/mL stock ofcarbenicillin are added.

288 W comprises 4.3 g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitaminsstock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, 0.10 g/Lpyridoxine HCl, and 0.40 g/L Glycine brought to volume with polished D-IH₂O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/Lmyo-inositol, 0.5 mg/L zeatin, and 60 g/L sucrose, which is then broughtto volume with polished D-I H₂O after adjusting to pH 5.6. Following,6.0 g/L of Ultrapure™ agar-agar (EM Science) is added and the medium issterilized and cooled. Subsequently, 1.0 mL/L of 0.1 mM abscisic acid;1.0 mg/L indoleacetic acid and 3.0 mg/L Bialaphos are added, along with2.0 mL of a 50 mg/mL stock of carbenicillin.

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycine brought tovolume with polished D-I H₂O), 0.1 g/L myo-inositol, and 40.0 g/Lsucrose (brought to volume with polished D-I H₂O after adjusting pH to5.6); and 6 g/L bacto-agar (added after bringing to volume with polishedD-I H₂O), sterilized and cooled to 60° C.

Example 5 Deletion Analysis of the RCc3 Promoter

The 1710 bp RCc3 promoter can be divided into 5 regions of 300-400 bpusing restriction endonuclease cleavage sites that naturally occur inthe promoter at the following positions (reading 3′ to 5′): BAMHI (0),BglII (232), BspMI (545), StuI (941), NheI (1354), and NotI (1710). Thisprovided an opportunity to generate four 5′ truncations of the promoter.Testing these truncations in plants may provide insight to regions ofthe promoter that play an important role in expression and the rootpreference of this promoter.

Digestion of the RCc3 promoter with NotI-NheI restriction endonucleasesresulted in the production of a 1354 bp promoter fragment, termed TR1.Digestion with NotI and StuI resulted in a 941 bp promoter fragment anddigestion with NotI and BspMI resulted in a 545 bp fragment,respectively termed TR2 and TR3. Cutting the promoter with NotI andBglII resulted in a promoter fragment of 232 bp, termed TR4. Each ofthese fragments was purified and ligated into an expression cassettethat resulted in each promoter fragment operably connected to theB-glucuronidase (GUS) gene.

Twenty-five transgenic maize events were regenerated for each truncationand grown under greenhouse conditions. Plants developed to V6/7 stagewhen leaf and root material was sampled for histochemical GUS staininganalysis. The plants were then grown to R1-R2 stage and sampled forstalk, tassel, and pollen. Results showed that none of the truncationsaffected expression in leaves, stalks, and pollen. No GUS expression wasdetected in any of these tissues (Table 3). Sheath tissue surroundingthe stalk was also checked for expression and none was detected (datanot shown). Expression in tassels was not detectable either, except for1 plant transformed with the 232 bp promoter fragment.

Expression in the root was not adversely affected by the truncations(Table 3). The overall expression pattern was generally retained, butthe expression level in the root tip which includes the meristematicregion and part of the elongation region decreased as the promoter wastruncated. The 232 bp promoter fragment, containing a putative TATA box,did not exhibit any root expression.

A number of motifs can be identified in the RCc3 promoter, such asROOTMOTIFTAPDX1 (ATATT) (data not shown). However, truncation of thepromoter suggests that the RCc3 promoter has sequences that providefunctional redundancy or that critical sequences for expression and rootpreference reside on the BspMI-BamH1 fragment. Root-preferred expressionis maintained to the BspMI cleavage site, but as the promoter istruncated to the BglII cleavage site the promoter is renderednon-functional

TABLE 3 Deletion Analysis Results V5-V6 R1-R2 Leaf Root Tip Root StalkTassel Pollen full-length 0 2 2 0 0 0 TR1 0 2 2 0 0 0 TR2 0 1 2 0 0 0TR3 0 1 2 0 0 0 TR4 0 0 0 0 0 0 Ubi-1 3 3 3 3 3 3 Untransformed 0 0 0 00 0 (negative control) Histochemical staining data is represented on a0-3 scale with the well characterized maize Ubi-1 promoter serving as areference point. The Ubi-1 promoter is a strong constitutive promoter innearly all tissues of maize.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

What is claimed is:
 1. An expression cassette comprising a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence comprising the sequence setforth in SEQ ID NO: 1; b) a nucleotide sequence comprising the plantpromoter sequence of the plasmid deposited as Patent Deposit No. NRRLB-50462; c) a nucleotide sequence comprising nucleotides 356 to 1710 ofSEQ ID NO: 1; d) a nucleotide sequence comprising nucleotides 769 to1710 of SEQ ID NO:1; and e) a nucleotide sequence comprising nucleotides1165 to 1710 of SEQ ID NO: 1; wherein said nucleotide sequence initiatestranscription in a plant cell, and a heterologous nucleotide sequence ofinterest operably linked to the nucleic acid molecule.
 2. A vectorcomprising the expression cassette of claim
 1. 3. A plant cellcomprising the expression cassette of claim
 1. 4. The plant cell ofclaim 3, wherein said expression cassette is stably integrated into thegenome of the plant cell.
 5. The plant cell of claim 3, wherein saidplant cell is from a monocot.
 6. The plant cell of claim 5, wherein saidmonocot is maize.
 7. The plant cell of claim 3, wherein said plant cellis from a dicot.
 8. A plant comprising the expression cassette ofclaim
 1. 9. The plant of claim 8, wherein said plant is a monocot. 10.The plant of claim 8, wherein said monocot is maize.
 11. The plant ofclaim 8, wherein said plant is a dicot.
 12. The plant of claim 8,wherein said expression cassette is stably incorporated into the genomeof the plant.
 13. A transgenic seed of the plant of claim 12, whereinthe seed comprises the expression cassette.
 14. The plant of claim 8,wherein the heterologous nucleotide sequence of interest comprises agene product that confers herbicide, salt, cold, drought, pathogen, orinsect resistance.
 15. A method for expressing a heterologous nucleotidesequence in a plant or a plant cell, said method comprising introducinginto the plant or the plant cell an expression cassette comprising apromoter operably linked to a heterologous nucleotide sequence ofinterest, wherein said promoter comprises a nucleotide sequence selectedfrom the group consisting of: a) a nucleotide sequence comprising thesequence set forth in SEQ ID NO: 1; b) a nucleotide sequence comprisingthe plant promoter sequence of the plasmid designated as Patent DepositNo. NRRL B-50462; c) a nucleotide sequence comprising nucleotides 356 to1710 of SEQ ID NO: 1; d) a nucleotide sequence comprising nucleotides769 to 1710 of SEQ ID NO: 1; and e) a nucleotide sequence comprisingnucleotides 1165 to 1710 of SEQ ID NO: 1; wherein said nucleotidesequence initiates transcription in said plant.
 16. The method of claim15, wherein the heterologous nucleotide sequence of interest comprises agene product that confers herbicide, salt, cold, drought, pathogen, orinsect resistance.
 17. The method of claim 15, wherein said heterologousnucleotide sequence of interest is expressed in a root-preferred manner.18. A method for expressing a heterologous nucleotide sequence in aroot-preferred manner in a plant, said method comprising introducinginto a plant cell an expression cassette, and regenerating a plant fromsaid plant cell, said plant having stably incorporated into its genomethe expression cassette, said expression cassette comprising a promoteroperably linked to a heterologous nucleotide sequence of interest,wherein said promoter comprises a nucleotide sequence selected from thegroup consisting of: a) a nucleotide sequence comprising the sequenceset forth in SEQ ID NO: 1; b) a nucleotide sequence comprising the plantpromoter sequence of the plasmid deposited as Patent Deposit No. NRRLB-50462; c) a nucleotide sequence comprising nucleotides 356 to 1710 ofSEQ ID NO: 1; d) a nucleotide sequence comprising nucleotides 769 to1710 of SEQ ID NO: 1; and e) a nucleotide sequence comprisingnucleotides 1165 to 1710 of SEQ ID NO: 1; wherein said nucleotidesequence initiates transcription in a plant root cell.
 19. The method ofclaim 18, wherein expression of said heterologous nucleotide sequence ofinterest alters the phenotype of said plant.